Neutrophils in Innate Immunity
Qin Wang, Ph.D.,
1Claire M. Doerschuk, M.D.,
1and Joseph P. Mizgerd, D.Sc.
2ABSTRACT
Neutrophils are an important component of innate immunity in the lungs. During bacterial pneumonia, neutrophils are recruited from the capillaries of the pulmonary cir-culation in the gas-exchanging regions of the lungs. This process requires the coordinated activation of many cells within the lungs, including neutrophils and capillary endothelial cells. Cellular activation during innate immune responses is mediated in part by tumor necrosis factor-alpha (TNF-a) and interleukin (IL)-1–initiated signaling through their receptors, activation of nuclear factor kappa B (NF-kB) and downstream gene transcrip-tion, endothelial cell signaling initiated by neutrophil adherence to intercellular adhesion molecule (ICAM)-1, and binding of leukocyte adhesion molecules to cellular and matrix ligands. These events are essential to effective host defense during pneumonia.
KEYWORDS:Neutrophils, pneumonia, cytokines, adhesion molecules, host defense
Objectives:Upon completion of this article, the reader should be able to: (1) summarize the roles of the cytokines tumor necrosis factor-alpha (TNF-a) and interleukin (IL)-1 and the transcription nuclear factor-kappa B (NF-kB) in innate immunity; and (2) describe neutrophil adhesion pathways and signaling induced by ligation of intercellular adhesion molecule (ICAM)-1.
Accreditation:The University of Michigan is accredited by the Accreditation Council for Continuing Medical Education to sponsor continuing medical education for physicians.
Credits:The University of Michigan designates this educational activity for a maximum of 1 category 1 credit toward the AMA Physician’s Recognition Award.
N
eutrophils are a critical arm of innate immu-nity in the lungs. Neutrophil recruitment in response to inflammatory stimuli within the lungs is regulated through complex parallel and sequential events. This review focuses primarily on neutrophil recruitment within the distal gas-exchanging regions of the lungs, where emigration occurs primarily through the pulmon-ary circulation rather than through the bronchial vessels. Most neutrophil emigration in the pulmonary circula-tion takes place in the capillaries rather than in the postcapillary venules of the systemic circulation. In re-sponse to many stimuli, initial events include the pro-duction of the cytokines, tumor necrosis factor alpha (TNF-a) and interleukin-1 (IL-1aand IL-1b).Activa-tion of the transcripActiva-tion factor nuclear factor-kappa B (NF-kB) is also often an early event in host defense, and TNF-a and IL-1 are important regulators of NF-kB activation. One of the genes regulated by NF-kB is intercellular adhesion molecule (ICAM)-1, an adhesion molecule that serves as the major endothelial cell receptor for CD11/CD18. When CD11/CD18 and ICAM-1 bind, intracellular signaling is in-duced in both cell types. CD11/CD18 is not always required for neutrophil emigration, and innate im-mune responses to organisms that induce common community-acquired pneumonias may result in neutro-phil emigration through CD11/CD18-independent pathways.
Pulmonary Host Defenses; Editor in Chief, Joseph P. Lynch, III, M.D.; Guest Editors, Steve Nelson, M.D., Theodore J. Standiford, M.D.
Seminars in Respiratory and Critical Care Medicine, volume 25, number 1, 2004. Address for correspondence and reprint requests: Claire M. Doerschuk, M.D., Rainbow Babies and Children’s Hospital, Room 787, 11100 Euclid Ave., Cleveland, OH 44106. E-mail: [email protected].
1Division of Integrative Biology, Department of Pediatrics, Rainbow Babies and Children’s Hospital and Case Western Reserve University,
Cleveland, Ohio;2Physiology Program, Harvard School of Public Health, Boston, Massachusetts. Copyright# 2004 by Thieme Medical
Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662. 1069-3424,p;2004,25,01,033,041,ftx,en;srm00274x.
This review examines these four aspects of neu-trophil recruitment during innate immune responses, TNF-a and IL-1–initiated signaling through their re-ceptors, activation of NF-kB and downstream gene transcription, endothelial cell signaling initiated by neu-trophil adherence to ICAM-1, and adhesion pathways mediating neutrophil emigration.
TNF-a AND IL-1
Neutrophil recruitment requires the coordinated expres-sion of genes for adheexpres-sion molecules and chemokines. During pneumonia, gene transcription is likely induced initially in cells in the air spaces whose pattern-recogni-tion receptors, such as Toll-like receptors,1 bind con-served microbial structures, such as lipopolysaccharide (LPS) or unmethylated CpG deoxyribonucleic acid (DNA). This initial wave of gene transcription also results in the expression of the early-response cytokines, TNF-aand IL-1. These cytokines induce the expression of adhesion molecules and chemokines in cells that have not been directly activated by microbe-derived li-gands.2,3In this way, proinflammatory signals originally elicited by microbes in the alveolar air spaces may be communicated to other cells in the lungs and to cells in extrapulmonary tissues.
TNF-ais elaborated as a membrane protein and released from the cell surface by TNF-a cleaving en-zyme.4 Two different receptors signal in response to TNF-a: TNF receptor 1 (TNFR1) and TNF re-ceptor 2 (TNFR2).5 Soluble TNF-a signals primarily through TNFR1, whereas TNFR2 responds preferen-tially to membrane-bound TNF-a.6,7 Both receptors activate NF-kB transcription factors and induce gene transcription.
IL-1a and IL-1b are also initially elaborated as cytosolic proteins that are released to the extracellular milieu subsequent to proteolytic processing by calpain, for IL-1a, or caspase-1, for IL-1b.8 The type I IL-1 receptor (IL1RI) binds to both IL-1a and IL-1b, functioning as a signaling receptor that activates NF-kB and induces gene expression.9In contrast, the type II IL-1 receptor (IL1RII) does not contain an intracellular signaling domain. By functioning as a decoy receptor, IL1RII decreases signaling from IL-1a and IL-1b.9 Thus signaling from IL-1a and IL-1b is mediated exclusively by IL1RI.
TNF-a, IL-1a, and IL-1bare expressed during pneumonia in the lungs of experimental animals and patients.10–17To test the hypothesis that TNF-aand/or IL-1a and b are essential to innate immune responses during pneumonia, the response toEscherichia coliwithin the lungs was compared in mice deficient in one or more of the receptors for these cytokines. Compared with WT (wild type) mice, TNF receptor–deficient mice lacking both TNFR1 and TNFR2 show no decrease in NF-kB
translocation, neutrophil emigration, or edema accumu-lation during E. coli pneumonia, suggesting that TNF receptors are not required for a full response to this organism when studied using mutant mice.18These mice do show an increase in the number ofE. coliremaining in the lung at this early time point of 6 hours, suggesting that TNF-amay facilitate the clearance of this organ-ism. Similarly, mice deficient in IL1R1 have no defect in neutrophil emigration or edema accumulation duringE. colipneumonia.19
Thus signaling through either TNF receptors or IL1R1 is not independently essential to these innate immune responses when examined using mutant mice. However, TNF-a and IL-1 induce overlapping signal-ing pathways and the expression of overlappsignal-ing sets of genes,20suggesting that responses toE. coliin the lungs may require signaling from either TNF-a or IL-1. In fact, mice deficient in both TNFR1 and IL1R1 have a defect in innate immunity toE. coli. Neutrophil emigration and edema accumulation are only half of that observed in the lungs of WT mice during E. coli
pneumonia.19 These studies suggest that TNFR1 and IL1RI have essential but overlapping roles in mediating innate immunity in the lungs. Furthermore, these data indicate that pathways exist for neutrophil emigration and edema accumulation independent of these two receptors. TNF-a signaling though TNFR2 is unlikely to contribute to these responses because TNFR1/ IL1R1–deficient mice given the soluble chimeric mole-cule TNFR: ImmunoglobulinG to antagonize TNF-a have no greater or additional defects.21 More likely, a portion of neutrophil emigration and edema accumula-tion elicited byE. coliin the lungs may occur through pathways that do not require these cytokines.
These studies indicate that TNF-a and IL-1 share functions contributing to neutrophil emigration and the accumulation of extravascular plasma during
E. coli pneumonia. Because neutrophils and plasma proteins are essential to combating bacterial infections, inhibiting these innate immune responses could com-promise host defenses. However, given that pneumonia is an important risk factor for acute lung injury and the acute respiratory distress syndrome, decreasing the in-flammatory response to microbes in the lungs may be protective for some patients. These studies suggest that the combined inhibition of signaling from TNF-aand IL-1 may be more effective than inhibition of TNF-aor IL-1 alone in limiting inflammation and injury during respiratory infection.
NF-jB
All three signaling receptors for TNF-a and IL-1, as well as the Toll-like receptors, which recognize con-served microbial structures, activate NF-kB transcrip-tion factors.26 NF-kB complexes include homo- or heterodimers of NF-kB proteins (RelA, RelB, c-Rel, p50, and p52). NF-kB complexes are maintained in the cytoplasm of most cells by their interactions with in-hibitor IkB proteins, preventing their nuclear import and mediating their nuclear export. Upon appropriate receptor stimulation, signaling pathways activate IkB kinases (IKK), which phosphorylate IkB proteins, re-sulting in their ubiquitination and proteasomal degrada-tion. NF-kB proteins contain nuclear localization sequences, and NF-kB complexes accumulate in the nucleus after liberation from IkB proteins. NF-kB complexes bind specific nucleic acid sequences (kB sites) in the DNA and recruit multiprotein complexes that modify histones, remodel chromatin, and direct the expression of nearby genes.27 Activation of NF-kB is sufficient to induce gene expression mediating neutro-phil recruitment because adenoviral expression of con-stitutively active IKK in lung cells in vivo in the absence of other events associated with inflammation causes expression of the chemokines KC and MIP-2 and the accumulation of neutrophils.28
NF-kB is activated during experimental lung infections and in patients with lung injury and modulates innate immune responses. For example,E. coliandE. coli
LPS in rodent lungs induce the rapid nuclear accumula-tion of the NF-kB complexes RelA/p50 and p50/ p50.17,18,29NF-kB complexes accumulate in the alveolar macrophages of patients with the acute respiratory dis-tress syndrome.30,31In particular, NF-kB RelA is essen-tial to induction of ICAM-1, IL-8, KC, and MIP-2 by LPS or cytokines in vitro,2,32,33and ICAM-1,34KC,22 and MIP-235 are required for neutrophil recruitment elicited by LPS in rodent lungs. Studies in vivo have been hampered because the targeted deletion of the gene
for RelA results in embryonic lethality.36The additional genetic deficiency of TNFR1 rescues RelA-deficient mice through the embryonic period,37 presumably due to essential roles for RelA in the expression of genes protective against TNFR1-induced cytotoxicity. How-ever, mice deficient in both TNFR1 and RelA (TNFR1/ RelA mutants) die within several weeks of birth.37The TNFR1/RelA mutants, in contrast to either the WT or TNFR1-deficient mice, demonstrate widespread evi-dence of infection, including pneumonia and bacteremia, and the life spans of TNFR1/RelA mice are prolonged by antibiotic treatment, indicating that RelA is required for effective host defenses and prevention of lethal bacterial infections.37
To determine whether RelA is essential to gene expression and neutrophil emigration in the lungs,E. coli
LPS was delivered intranasally to TNFR1/RelA mutant mice at 3 to 5 days of age. The increase in expression of molecules mediating neutrophil emigration, including ICAM-1, KC, and MIP-2, is significantly less in the lungs of TNFR1/RelA mice compared with WT or TNFR1-deficient mice.37Furthermore, neutrophil emi-gration is compromised in TNFR1/RelA mutant mice compared with WT or TNFR1- deficient mice.37Thus, RelA mediates the LPS-induced expression of chemo-kines and adhesion molecules required for neutrophil emigration in the lungs.
The cells in which RelA-induced gene expression is particularly important for neutrophil emigration have not been conclusively identified. Reconstitution of lethally irradiated WT mice with RelA-deficient hematopoietic stem cells results in WT mice with RelA-deficient leukocytes, including alveolar macro-phages and circulating neutrophils.37 Compared with WT mice with WT leukocytes, neutrophil emigration elicited by LPS in the lungs is not decreased in the lungs of WT mice with leukocytes deficient in RelA, TNFR1, or both.37 Comparing these results with the studies of mice deficient in TNFR1 and RelA in all cells suggests that gene induction by RelA in nonhematopoietic cells, such as epithelial and endothelial cells of the lung, is critical to neutrophil recruitment.
the LPS-induced degradation of IkB-a and IkB-b. Studies to determine the functions of these IkB have demonstrated that IkB-b deficiency did not affect the nuclear accumulation of NF-kB complexes, neutrophil recruitment to alveolar air spaces, or pulmonary edema,29 suggesting IkB-bproteins do not possess unique proper-ties essential to NF-kB translocation or to acute inflam-mation induced by E. coli LPS in the lungs. Studies examining IkB-aare not yet feasible because deficiency of this molecule results in uncontrolled inflammation and perinatal lethality.38
IkB proteins are only one mechanism by which cells inhibit RelA. Other endogenous means for inhibit-ing gene induction by RelA include p50/p50 homodi-mers,39–41 steroid hormones,42,43 cyclopentenone prostaglandins,44 histone deacetylases,45,46 and Twist transcription factors.47 Mimicking these inhibitory pathways with pharmacological agents may be candidate approaches for patients with excessive lung inflamma-tion. Furthermore, host variation in these molecular pathways, resulting from genetic polymorphisms and previous or current environmental stresses, may predis-pose subpopulations to acute lung injury during infec-tion. The role of RelA in mediating the LPS-induced expression of proinflammatory genes and amplifying neutrophil recruitment suggests that limiting RelA ac-tivity may be important to protecting the lungs from injury.
ICAM-1–DEPENDENT SIGNALING INTO PULMONARY MICROVASCULAR ENDOTHELIAL CELLS DURING NEUTROPHIL ADHESION
ICAM-1 is an adhesion molecule expressed by endothe-lial and other cells. In many pulmonary inflammatory responses, such as those induced byE. coliLPS or TNF-a, ICAM-1 expression is increased on the luminal sur-face of pulmonary capillary endothelium.48,49Its expres-sion is regulated by NF-kB.2,37The interaction between neutrophil b2 integrins and endothelial cell (EC) ICAM-1 often mediates the adhesion of neutrophils to pulmonary capillary ECs, a process that is usually a prerequisite for subsequent neutrophil migration across the endothelium and into the alveolar space during pulmonary inflammation.50 Blockade of ICAM-1 ex-pression in the lungs by antisense oligonucleotides or treatment with anti–ICAM-1 antibodies largely pre-vents neutrophil sequestration and emigration into the alveolar space induced by many inflammatory sti-muli,51–53 demonstrating an indispensable role for ICAM-1 in these responses.
Recent studies using cultured ECs have provided evidence that ICAM-1, in addition to its role as an adhesion molecule for leukocytes, functions as a signal-ing molecule when ligated by leukocytes, antibodies, or
fibrinogen.54–59These studies led to the hypothesis that ICAM-1–induced signaling cascades and downstream events in pulmonary capillary ECs may play important roles in modulating neutrophil migration along or across ECs. Support for this concept is provided by studies of Sans and colleagues,60 who demonstrated that in a reconstituted cell line, expression of ICAM-1 without its cytoplasmic domain required for signaling prevents neutrophil transmigration without inhibiting neutrophil adhesion. Using cultured human and rat pulmonary microvascular ECs, we have recently shown that ICAM-1 ligation induces a cascade of signaling events into ECs, resulting in cytoskeletal changes of ECs.61–64 Inhibition of signaling events in ECs prevents neutro-phil migration toward EC borders, the preferred site of neutrophil emigration across pulmonary capillary ECs in vivo.65,66 ICAM-1–initiated signaling cascades are the focus of the remainder of this section.
ICAM-1–induced changes in the F-actin cytoskeleton do not occur when only the primary antibody is bound but rather require the addition of a secondary antibody, suggesting that cross-linking ICAM-1 (i.e., bringing more ICAM-1 molecules closer together) is necessary for transducing signaling events into ECs.
Ongoing studies are beginning to delineate the signaling pathways initiated by ICAM-1 that result in these changes in the F-actin cytoskeleton following neutrophil adherence. Cross-linking ICAM-1 with antibodies induces redistribution of ICAM-1 into ag-gregates on the surface of human pulmonary microvas-cular ECs.61,64In cultured human umbilical vein ECs, clustering of ICAM-1 and its association with actin-binding proteins such as ezrin (a membrane cytoskeletal linker),a-actinin, and vinculin in response to ligation of ICAM-1 by antibodies or adherence of leukocytes have been observed.54,70 These changes in ICAM-1 require phosphoinositides and Rho, and are thought to play important roles in mediating firm adhesion of leuko-cytes.54,70The role of ezrin, phosphotidylinositols, and Rho in ICAM-1–initiated signaling within human pul-monary microvascular ECs remains to be determined.
Downstream signaling events following ICAM-1 ligation and clustering on pulmonary microvascular ECs are depicted in Fig. 1. They include activation of xanthine oxidase and generation of reactive oxygen species (ROS) by this enzyme in ECs.61,64 Production of ROS in turn leads to activation of p38 mitogen-activated protein kinase (MAPK) and subsequent phos-phorylation of heat shock protein 27 (hsp27), an actin-binding protein that may modulate F-actin polymeriza-tion upon phosphorylapolymeriza-tion.62,64,71Pretreatment of ECs with SB203580, an inhibitor of p38 MAPK, prevents the changes in the F-actin cytoskeleton in ECs induced by neutrophils or cross-linking ICAM-1, indicating that p38 MAPK is required for the cytoskeletal changes initiated by ICAM-1 ligation.62
These studies led to the hypothesis that during neutrophil adherence to TNF-a–treated pulmonary mi-crovascular ECs, ICAM-1–initiated signaling events and cytoskeletal changes may modulate neutrophil mi-gration along ECs to reach EC borders. Our studies demonstrate that there is a time-dependent increase in the percentage of neutrophils present at EC borders after addition of neutrophils to TNF-a–treated ECs, indicat-ing that neutrophils migrate toward EC borders.63 Pre-treatment of ECs with an inhibitor of p38 attenuates neutrophil migration toward EC borders.62 These data suggest that activation of p38 and phosphorylation of its downstream substrates in ECs play important roles in mediating neutrophil migration toward EC borders, where neutrophil transmigration occurs.
Neutrophil emigration occurs within the micro-vasculature, primarily from the pulmonary capillaries during inflammation in the lung parenchyma.50
Endothelial cells of the pulmonary vasculature may have specialized responses to neutrophil adhesion. For example, comparison of cultured pulmonary arterial ECs (PAECs) and pulmonary microvascular ECs (PMECs) isolated from rats show that adherence of rat neutrophils induces an increase in F-actin staining and the stiffening response of TNF-a–pretreated rat PMECs, but not rat PAECs.64 This difference between ECs isolated from these two sites is not due to differences in the expression of ICAM-1 following TNF-atreatment or in ICAM-1 clustering in response to cross-linking antibodies. How-ever, ICAM-1–dependent activation of p38 MAPK occurs in TNF-a–pretreated PMECs, but not PAECs. This activation of p38 in PMECs is prevented by allopurinol, an inhibitor of xanthine oxidase (see Fig. 1). Measurement of xanthine oxidase activity reveals that this enzyme’s activity in PMECs is increased by neutrophil adhesion. In contrast, PAECs demonstrate a much higher basal level of activity of this oxidase, and no detectable increase is induced by neutrophil adherence.
Taken together, these data suggest that differences between rat PMECs and PAECs in their cytoskeletal responses to neutrophil adherence are due to differences in ICAM-1–initiated signaling pathways. The differ-ence in signaling seems to lie between ICAM-1 cluster-ing (which occurs in both cell types) and p38 activation, perhaps in the activation of xanthine oxidase.
Whether these ICAM-1–initiated signaling events and actin cytoskeletal changes in ECs during neutrophil adherence are required for neutrophil emigra-tion into the alveolar space during inflammatory pro-cesses in vivo remains to be determined. Dynamic changes in actin cytoskeletal organization do occur and appear essential for neutrophil emigration during in-flammatory responses in vivo. For instance, intratracheal instillation of phalloidin, which prevents F-actin rear-rangement in lung capillary ECs and epithelial cells, inhibits neutrophil emigration into the alveolar spaces by 83% in response to Streptococcus pneumoniae.72In addi-tion, phalloidin instillation attenuates neutrophil emi-gration and edema formation in acid aspiration lung injury.73 These studies suggest that changes in the cytoskeleton in ECs and epithelial cells are required for neutrophil emigration during acute pulmonary in-flammation. Generation of genetically altered animal models such as animals expressing a truncated form of ICAM-1 without its cytoplasmic domain required for signaling will help elucidate the physiological signifi-cance of ICAM-1–initiated signaling events in mediat-ing neutrophil emigration in lung inflammation in vivo.
NEUTROPHIL EMIGRATION
Although adhesion mediated by CD11/CD18 and ICAM-1 ligation is required for neutrophil emigration in response to some stimuli (including E. coli, E. coli
endotoxin, Pseudomonas aeruginosa, IL-1, IgG immune complexes), the CD11/CD18 adhesion complex is not required in response to others (includingS. pneumoniae,
Staphylococcus aureus, Group B Streptococcus, C5a, KC, hyperoxia50). The selection of CD11/CD18–dependent or –independent pathways appears to be determined by which cytokines are produced very early in host defense toward these stimuli.50 Although TNF-a and IL-1 appear important in initiating CD11/CD18–mediated adhesion, other cytokines such as interferon gamma (IFN-g) appear important in regulating the CD11/ CD18–independent pathway.74 CD11/CD18–inde-pendent adhesion is not mediated by members of the selectin family, VLA-4 (very late antigen-4), or PECAM-1 (platelet–endothelial cell adhesion mole-cule-1).50,75,76 Presently, the molecules that mediate adhesion when CD11/CD18 is not required are not known, and whether neutrophil–endothelial cell adhe-sion molecules in the traditional sense are needed is also not clear.
Following adhesion and transendothelial migra-tion, neutrophils pass through slitlike holes in the en-dothelial basal lamina into the connective tissue matrix within the thick side of the capillary loop.65,66 The cellular processes of fibroblasts within this matrix extend from these holes to similar holes within the basal lamina of alveolar epithelial cells.65,66Neutrophils are often in contact with these fibroblasts as they crawl toward the alveolar spaces, suggesting that they use these cells as a track. Neutrophils prefer to pass between Type I and Type II cells, rather than between Type I/Type I borders.65,66 CD44, the receptor for hyaluronic acid and other connective tissue molecules, andb1 integrins appear important in neutrophil emigration toward some stimuli.77,78The molecular signals regulating neutrophil transit following transendothelial cell migration are only beginning to be elucidated.
SUMMARY
Host defense and innate immunity are complex process-es that often require recruitment of neutrophils. Produc-tion of TNF-a and IL-1, activation of NF-kB, and subsequent gene expression are important steps in neu-trophil recruitment early in the response to many stimuli. Neutrophil–endothelial cell interactions through CD11/CD18-mediated adhesion to ICAM-1 are also often but not always critical and depend upon the stimulus. Both the adhesive function of these molecules and the signaling cascades that are initiated within both cell types may be important in modulating neutrophil recruitment and their subsequent ability to combat infection. Our understanding is incomplete at this time, and our ability to modulate innate immunity therapeutically will be greatly enhanced by a clearer understanding of the multiple pathways through which the host responds to stimuli within the lungs.
FUNDING
Supported by: NIH HL 48160, 52466, and 68153, a Parker B. Francis Fellowship from the Francis Families Foundation (Q.W.), a Research Grant from the American Lung Association (Q.W.), and a Clinical Investigator Award in Translational Research (C.M.D).
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